title: Analysis of protein-ligand interactions by fluorescence polarization.
abstract: Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a nondisruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP(3)) to N-terminal fragments of IP(3) receptors can be characterized at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real time without the use of radioactive materials, it is nondestructive and, with appropriate care, it can resolve ΔH° and ΔS°. The first part of the protocol, protein preparation, may take several weeks, whereas the FP measurements, once they have been optimized, would normally take 1-6 h.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Rossi
authors.forename: Colin W
authors.initials: CW
authors.lastname: Taylor
mesh_terms: D000465:Algorithms; D000818:Animals; D001667:Binding, Competitive; D005454:Fluorescence Polarization / Q000379:methods*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D051381:Rats; D013816:Thermodynamics
chemical_list: D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D015544:Inositol 1,4,5-Trisphosphate